IP3-ACTIVATED CA2+ CHANNELS IN THE PLASMA-MEMBRANE OF CULTURED VASCULAR ENDOTHELIAL-CELLS

Although it is clear that D-myo-inositol 1,4,5-trisphosphate (IP3) plays an important role in the activation of Ca2+ influx, the mechanisms by which this occurs remain controversial. In an attempt to determine the role of IP3 in the activation of Ca2+ influx, patch-clamp single-channel experiments in the cell-attached, inside-out, and outside-out configurations were performed on cultured bovine aortic endothelial cells (BAEC). The results presented indicate that both IP3 and intracellular Ca2+ can modulate the activity of a Ca2+-selective channel found in the plasma membrane of these cells. Addition of 10 mu M IP3 increased channel open probability (P-o) from a control value of 0.12 +/- 0.05 to 0.7 +/- 0.13 at a constant intracellular Ca2+ of 1 nM in excised inside-out patches. D-Myo-inositol 1,3,4,5-tetrakisphosphate at 50 mu M was ineffective in altering channel P-o. Channel activity declined after similar to 2 min in the continuous presence of IP3. Three to four minutes after addition of IP3, channel P-o was reduced from 0.7 +/- 0.2 to 0.2 +/- 0.1, indicating that an additional regulator might be required to maintain channel activity in excised patches. The channel was reversibly blocked by application of 1 mu g/ml heparin to the intracellular side of inside-out patches. This Ca2+-selective channel is indistinguishable from the depletion-activated Ca2+ channel we have previously described in BAEC.